Structural characterization of arachidonyl radicals formed by prostaglandin H synthase-2 and prostaglandin H synthase-1 reconstituted with mangano protoporphyrin IX.
A tyrosyl radical generated in the peroxidase cycle of prostaglandin H synthase-1 (PGHS-1) can serve as the initial oxidant for arachidonic acid (AA) in the cyclooxygenase reaction. Peroxides also induce radical formation in prostaglandin H synthase-2 (PGHS-2) and in PGHS-1 reconstituted with mangano protoporphyrin IX (MnPGHS-1), but the EPR spectra of these radicals are distinct from the initial tyrosyl radical in PGHS-1. We have examined the ability of the radicals in PGHS-2 and MnPGHS-1 to oxidize AA, using single-turnover EPR studies. One wide singlet tyrosyl radical with an overall EPR line width of 29-31 gauss (G) was generated by reaction of PGHS-2 with ethyl hydroperoxide. Anaerobic addition of AA to PGHS-2 immediately after formation of this radical led to its disappearance and emergence of an AA radical (AA.) with a 7-line EPR, substantiated by experiments using octadeuterated AA. Subsequent addition of oxygen resulted in regeneration of the tyrosyl radical. In contrast, the peroxide-generated radical (a 21G narrow singlet) in a Y371F PGHS-2 mutant lacking cyclooxygenase activity failed to react with AA. The peroxide-generated radical in MnPGHS-1 exhibited a line width of 36-38G, but was also able to convert AA to an AA. with an EPR spectrum similar to that found with PGHS-2. These results indicate that the peroxide-generated radicals in PGHS-2 and MnPGHS-1 can each serve as immediate oxidants of AA to form the same carbon-centered fatty acid radical that subsequently reacts with oxygen to form a hydroperoxide. The EPR data for the AA-derived radical formed by PGHS-2 and MnPGHS-1 could be accounted for by a planar pentadienyl radical with two strongly interacting beta-protons at C10 of AA. These results support a functional role for peroxide-generated radicals in cyclooxygenase catalysis by both PGHS isoforms and provide important structural characterization of the carbon-centered AA..
Tsai
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Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA. atsai@heart.med.uth.tmc.edu
Palmer
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Xiao
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Swinney
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Kulmacz
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RJ
D016718:Arachidonic Acid / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry; D007527:Isoenzymes / Q000378:metabolism; D015289:Leukotrienes / Q000378:metabolism; D008054:Lipid Peroxides / Q000378:metabolism; D008968:Molecular Conformation; D009154:Mutation / Q000235:genetics; D010100:Oxygen / Q000378:metabolism; D010544:Peroxidases / Q000378:metabolism; D010545:Peroxides / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism*; D011524:Protoporphyrins / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry
D005609:Free Radicals; D007527:Isoenzymes; D015289:Leukotrienes; D008054:Lipid Peroxides; D010545:Peroxides; D011524:Protoporphyrins; C025577:ethyl hydroperoxide; C038654:manganese protoporphyrin; D016718:Arachidonic Acid; D014443:Tyrosine; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; D010544:Peroxidases; D011451:Prostaglandin-Endoperoxide Synthases; D010100:Oxygen